Method and apparatus for rapid determination of blood sedimentation rate

ABSTRACT

An apparatus and method for rapid determination of erythrocyte sedimentation rates for a blood specimen (29) which can be linearly transposed to Westergren sedimentation rates. The method includes the steps of inducing accelerated rouleaux formation in the specimen (29) in an amount sufficient to begin settling at substantially the decantation rate for the specimen. In one embodiment a structure (27) which produces a very thin cross-sectional region (37) of the specimen (29) inside the lumen (23) of a specimen container (21) is provided to accelerate rouleaux formation. In an alternative embodiment (120), accelerated rouleaux formation is accomplished using a centrifuge (122). A third embodiment employs a movable rod (223) mounted inside the specimen tube (221) to induce accelerated rouleaux formation. All embodiments of the process next employ gravity settling the specimen in a near horizontal oriented container (21, 121, 221). Thereafter, the amount of settling occurring is determined. A sealed specimen container (21, 121, 221) which permits thorough mixing of blood in a very small diameter container or tube by a tilting action and accompanying liquid movement past a moving bubble within the tube lumen for use in performing the method also is provided.

This is a division of application Ser. No. 08/718,637 filed Sep. 17,1996, which is a division of application Ser. No. 08/270,681, filed Jul.12, 1994 which is now U.S. Pat. No. 5,594,164 .

TECHNICAL FIELD

The present invention relates, in general, to a method and apparatus fordetermining the settling rate of solids in a liquid-solid mixture, andmore particularly, relates to methods and apparatus for determining theerythrocyte or red blood cell sedimentation rate in whole blood.

BACKGROUND ART

The rate at which erythrocytes or red blood cells settle through bloodplasma in a whole blood specimen has long been the subject of medicalstudy. It has been found that the sedimentation rate of blood can besignificantly increased by a wide range of inflammatory conditions anddiseases. Various attempts have been made to automate bloodsedimentation apparatus and to correlate settling or sedimentation ratesand patterns to inflammatory conditions.

The original laboratory studies, however, are still regarded as thestandards. More particularly, there is a Wintrobe sedimentation methodand a Westergren sedimentation method. The Westergren method is mostwidely used and employs a 300 millimeter long settling tube with thelower 200 millimeters being graduated. The tube is filled to the 200millimeter mark with approximately 0.8 milliliter of blood and 0.2milliliters of anticoagulant diluent which is allowed to gravity settleover a one or a two hour period. The amount of settling in a one hourperiod in a westergren settling tube is generally regarded as thestandard for blood sedimentation rate.

In any blood sedimentation study the specimen is first thoroughly mixedso that the erythrocytes are evenly distributed throughout the specimen.In the Westergren method, after mixing, the 300 millimeter tube isbrought to a vertical orientation for gravity settling and the settlingclock started. After one hour the amount of settling which has occurred,as determined by the distance that the interface between the plasma andthe erythrocytes has traveled downward, is measured.

Various attempts have been made to automate the Westergren settlingprocess. U.S. Pat. No. 4,041,502 to Williams, et al., for example,discloses an automated sedimentation measuring system in whichmeasurements are taken every 15 seconds for one hour (or two hours) anda blood sedimentation curve is produced as a result of thesemeasurements. U.S. Pat. No. 4,848,900 to Kuo, et al. is a similar bloodsedimentation automated system in which a blood sedimentation curve isalso generated over a one hour period.

While the amount of sedimentation varies for each specimen, asinfluenced by inflammatory conditions, the general shape of bloodsedimentation curves is quite similar as a result of the settlingphenomena which are operative. Most sedimentation curves, therefore,have three phases which can be clearly identified. First, there is a"lag phase" in which settling is very slow and gradual. Next comes a"decantation phase" in which rapid, virtually linear, settling occurs.Finally, there is a "syneresis phase" in which the rate of settlinggreatly slows towards the end of the one hour settling period.

While the erythrocytes are more dense than the plasma, they are smalland thus have such a high surface area relative to volume that they donot readily sediment through plasma as single cells. In the initial orlag phase, therefore, the erythrocytes must come in contact with eachother to group together in clumps or clusters known as "rouleaux." Oncea sufficient number of erythrocytes have grouped in rouleaux, therouleaux will begin to sediment through the blood plasma toward thebottom of the sedimentation tube. Thus, the initial sedimentation or lagphase may take 5 to 15 minutes for sufficient rouleaux clusters to formto enter the more rapid decantation phase. The lag phase portion of asedimentation vs. time curve, therefore, is relatively flat andtypically shows little sedimentation or movement of theplasma-erythrocyte separation interface.

As the erythrocytes in rouleaux settle, they contact other erythrocyteswhich adhere and decrease the surface are a to volume ratio and hencethe drag on the sedimenting rouleaux. Additionally, however, the plasmaat the bottom of the sedimentation tube must rise or be displacedupwardly by a volume equal to the sedimenting erythrocytes. The settlingprocess, therefore, involves both downward migration or sedimentation ofthe more dense erythrocytes in rouleaux and upward migration of thelighter plasma through the downwardly migrating rouleaux. Thesedimentation rate increases significantly and is fairly linear in thedecantation phase or the mid-range of the sedimentation process.

Toward the end of the sedimentation process, however, the erythrocytesbegin to pack more tightly at the bottom of the tube. This narrows thepathways for plasma to upwardly migrate to the plasma-erythrocyteseparation boundary or interface. The plasma, therefore, has a moredifficult time escaping from between the red cells in rouleaux as thepacking density or hematocrit rises. Sedimentation, therefore, againslows in this last or syneresis phase of sedimentation.

Various attempts have been made to devise methods and apparatus foraccelerating the determination of erythrocyte sedimentation rates. In myU.S. Pat. No. 3,824,841, for example, a sedimentation method isdisclosed in which specimens are centrifuged in vertically orientedsettling tubes, with the tubes periodically rotated about theirlongitudinal axis. The erythrocytes seesaw back and forth across thetube and downwardly under a combination of gravity and centrifugalforces. The sedimentation time using this process is reduced from onehour to about three minutes, and the sedimentation rates measured usingthis process and apparatus can be related to the Westergren method usingnon-linear regression algorithms. This apparatus and method, however,have drawbacks in the form of the complexity of the apparatus, as wellas the need to use non-linear regression algorithms.

Sedimentation studies also have been undertaken in shorter tubes andparticularly 100 millimeter settling tubes. The problem with thistechnique is that the final or syneresis phase, in which the hematocritis rapidly rising, occurs earlier, again requiring non-linear algorithmsfor correlation to Westergren sedimentation results.

Additionally, accelerated blood sedimentation has been measured usingtilted or inclined sedimentation tubes instead of vertically orientedtubes. Using an inclined tube (a tube 100-200 millimeters long and 2.5millimeters in diameter inclined at about 30 or 45 degrees fromvertical) settling rates can be measured after only 20 minutes ofsettling. The settling rate which is determined using such apparatus,however, is not a true Westergren sedimentation rate because once againthe relationship between the hematocrit rise and the entrapment ofplasma is altered resulting in a nonlinear relationship between the testmethod. Thus, the settling is non-linear and the measured rates must berelated to Westergren rates by non-linear correlation algorithms.Tilting of the tube reduces the settling time by about two-thirds butbecause there is in this method no shortening of the lag phase, 20minutes is still required and a non-linear correlation to Westergrenrates is also necessary.

In general, the use of non-linear algorithms becomes less reliable inrelating results to Westergren sedimentation rates as the sedimentationrate increases. High sedimentation rates usually indicate the presenceof inflammatory conditions. Thus, the non-linear effects induced byrapid sedimentation tend to decrease the correlation accuracy toWestergren rates for specimens which are most affected by disease andother conditions sought to be discovered or analyzed by thesedimentation process.

Accordingly, it is an object of the present invention to provide anapparatus and method for erythrocyte blood sedimentation which can berapidly accomplished and yet is capable of high correlation by lineartransposition to Westergren sedimentation rates.

Another object of the present invention is to provide an erythrocytesedimentation method and apparatus which can be used repeatedly on thesame specimen to rapidly determine and verify the blood sedimentationrate.

Another object of the present invention is to provide a bloodsedimentation apparatus and method in which the whole blood specimen,from the moment of venipuncture, remains in a sealed container.

Still a further object of the present invention is to provide a bloodsedimentation apparatus and method in which smaller blood specimens arerequired and mixing of the blood specimen can be readily accomplished invery small volumes.

Another object of the present invention is to provide an improvedspecimen container for use with processes requiring mixing of smallliquid volumes.

Still another object of the blood sedimentation apparatus and method ofthe present invention is to provide a sedimentation process havingincreased accuracy, relatively low cost, and suitability forsemi-automated use by relatively unskilled paramedical personnel.

The blood sedimentation method and apparatus of the present inventionhave other objects and features of advantage which will become apparentfrom, and are set forth in more detail in, the accompanying drawing andfollowing Best Mode Of Carrying Out The Invention.

DISCLOSURE OF INVENTION

The method of the present invention provides a process for accelerateddetermination of erythrocyte sedimentation in whole blood which can becorrelated with a very high confidence level to Westergren settlingrates using linear data transposition. The present method greatlyaccelerates the lag phase by using one of several techniques,accomplishes the gravity decantation phase rapidly by orienting thespecimen container in a manner reducing the time required for settling,and essentially eliminates the syneresis phase, again by orienting thespecimen container to avoid plasma trapping as hematocrit rises.

The method for accelerated determination of erythrocyte sedimentation ofthe present invention comprises, briefly, inducing rouleaux formation ina time period substantially less than the Westergren lag phase timeperiod for the same specimen and in an amount sufficient for thespecimen to enter the decantation phase. In the preferred form of lagphase acceleration, a portion of the specimen is formed into a very thincross-section in the specimen container through which a fluid current isinduced to flow so that contact between individual erythrocytes isenhanced and rouleaux formation in such portion occurs rapidly. Thisthin cross-sectional portion of the container is advantageously providedby a rod positioned inside the lumen of a tubular container andextending beyond the top surface of the specimen to cause a veil ofspecimen material to form by capillary attraction between the rod andcontainer. Moreover, the very thin cross-sectional portion of thespecimen is oriented preferably at about 20 to about 30 degrees fromhorizontal for gravitational movement of the rapidly formed rouleauxdown through the specimen to seed the specimen and commence thedecantation phase.

In one alternative form of the present process, lag phase accelerationis accomplished by centrifugating the specimen, rather than creating acapillary veil, to form rouleaux rapidly. In still a further alternativeprocess, a confined cross-sectional portion of the specimen container isused and combined with a change of the specimen container configurationto accelerate the lag phase. This form of lag phase acceleration isaccomplished by holding a rod, for example magnetically, against anupper side of a horizontally oriented container lumen and then releasingthe rod to gravitate down through the specimen to a lower side of thelumen.

The present process further includes the step of gravity settling theerythrocytes, after rouleaux formation, with the specimen containeroriented to substantially reduce the time required for the decantationphase below that which would be required in the Westergren process forthe same specimen. Such gravity settling is accomplished, for example,by maintaining the specimen container oriented at 30 degrees or less tothe horizon. This orientation, together with the presence of the rod onthe upper side of the tube lumen, provides two protected channelsthrough which plasma can escape thus significantly shortening thesyneresis phase of sedimentation. The method of the present inventionincludes as a final step determining the amount of sedimentation whichhas occurred in the specimen, preferably by reorienting the specimencontainer from its near horizontal orientation for gravity settling to anear vertical orientation for sedimentation measurement.

The apparatus for accelerated determination of erythrocyte sedimentationrate is comprised, briefly, of an elongated specimen tube having a lumenformed to contain a blood specimen therein and preferably including avery thin cross-sectional portion. The thin cross-sectional portion ofthe lumen advantageously can be provided by mounting a rod, adhesivelyor magnetically, in the specimen tube next to a wall of the tube and ina position to extend above a top surface of the specimen so that a veilof blood will form between the rod and tube wall.

The present apparatus also includes specimen tube orienting assemblywhich can hold the specimen tube in a manner orienting it foraccelerated rouleaux formation, for example at about 30 degrees from ahorizontal plane. The orienting assembly also orients the specimen tubefor accelerated gravity settling during the decantation phase, again atabout 30 degrees or less to a horizontal plane. Preferably the orientingassembly is further formed to enable manipulation of the specimen tubeto enable specimen mixing and reorientation from a gravity settlingorientation to a sedimentation determination orientation.

In a first alternative embodiment of the apparatus the orientingassembly is also formed for centrifugation of the specimen tube, and ina second alternative embodiment the orientation assembly is formed toselectively hold and release a rod mounted in the specimen tube.

In another aspect of the present invention, a specimen container formixing small volumes of liquid, such as blood specimens, is provided.The specimen container has an elongated bore with an elongated membermounted therein to define a resulting elongated lumen with a channelportion of the cross sectional area of the lumen being formed to besufficiently thin in transverse cross section that a liquid-mixing gasbubble cannot enter this channel portion of the lumen and the liquid canflow along the channel.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top perspective view an erythrocyte sedimentation apparatusconstructed in accordance with the present invention.

FIG. 2 is a top perspective view corresponding to FIG. 1 with the bloodspecimen tubes oriented for accelerated rouleaux formation and gravitysettling.

FIG. 3 is an enlarged, top plan view in cross section of the apparatustaken substantially along line 3--3 in FIG. 1.

FIG. 4 is a greatly enlarged, side elevation view, in cross section, ofa specimen container tube constructed in accordance with the presentinvention.

FIG. 5 is an end view, in cross section, taken substantially along theplane of line 5--5 in FIG. 4.

FIG. 6 is a graph of sedimentation rate data using the apparatus ofFIGS. 1-5, as compared to Westergren sedimentation data for modifiedblood specimens from the same patients.

FIG. 7 is a top perspective view of an alternative embodiment of anerythrocyte sedimentation apparatus constructed in accordance with thepresent invention.

FIG. 8 is an enlarged, side elevation view, in cross section, of aspecimen container designed for use with the apparatus of FIG. 7.

FIG. 9 is a further enlarged, cross sectional view taken substantiallyalong the plane of line 9--9 in FIG. 8.

FIG. 9A is a cross sectional view corresponding to FIG. 9 of analternative embodiment of the specimen collection tube of FIG. 8.

FIG. 9B is a cross sectional view corresponding to FIG. 9 of anotheralternative embodiment of the specimen collection tube of FIG. 8.

FIG. 10 is a schematic representation of the specimen tube of FIG. 8after drawing of a specimen.

FIG. 11 is a schematic representation of the specimen tube of FIG. 8during mixing.

FIG. 12 is a schematic representation of the specimen tube of FIG. 8during centrifuging.

FIG. 13 is a schematic representation of the specimen tube of FIG. 2 atthe start of gravity settling.

FIG. 13A is an enlarged, cross sectional view of the specimen tube atthe start of settling.

FIG. 13B is an enlarged, cross sectional view of the specimen tube aftersignificant settling has occurred.

FIG. 14 is a schematic representation of the specimen tube of FIG. 8during the step of determining the amount of sedimentation.

FIG. 15 is a graph of sedimentation rate data using the apparatus ofFIGS. 7 and 8, as compared to Westergren sedimentation data forunmodified blood specimens from the same patients.

FIG. 16 is a graph of sedimentation rate data using the apparatus ofFIGS. 7 and 8, as compared to Westergren sedimentation data for modifiedblood specimens from the same patients.

FIG. 17 is a side elevation view, in cross section of an alternativeembodiment of a specimen container constructed in accordance with thepresent invention.

BEST MODE OF CARRYING OUT THE INVENTION

The method and apparatus of the present invention achieve rapiderythrocyte sedimentation results which can be linearly related toWestergren sedimentation results. Essentially two key principles areemployed to greatly accelerate the time required for sedimentation.First, the lag phase of the conventional Westergren sedimentation methodis accelerated by inducing rouleaux formation in the specimen rapidly,until there is sufficient rouleaux to cause erythrocyte settling atsubstantially the decantation rate for the specimen. Next, the specimenis gravity settled in a container formed and oriented so that theerythrocytes can settle through upwardly migrating plasma in a mannerwhich is substantially unimpeded by the increasing hematocrit.

The method and apparatus of the present invention allow a blood specimensedimentation to be accelerated across the lag phase, gravity settledthrough the decantation phase in a much shorter period of time, andsettling in the syneresis phase to essentially be eliminated. The resultis that sedimentation data that can be linearly transposed to Westergrendata can be obtained in five minutes or less.

Lag Phase Acceleration

The first step of the present process is to accelerate sedimentation bygreatly reducing the time which would normally be required for thespecimen to pass through the lag phase and begin the linear decantationphase. Several techniques for acceleration of blood sedimentationthrough the lag phase have been discovered and will be described herein,but the preferred technique can be described by reference to a specimentube which is particularly well suited for use in the method of thepresent and is shown in FIGS. 4 and 5. Essentially the same tube also isshown in FIGS. 8 and 9.

As will be seen from FIGS. 4 and 5, elongated specimen tube or container21 is formed with a tube wall 22 which defines a tube lumen 23. Lumen 23terminates in an open end 24 having a rubber stopper or other endclosure member 26 mounted therein. As will be described in greaterdetail hereinafter, specimen tube 21 advantageously can be a partiallyevacuated, blood specimen tube having a length of about 110 millimetersand a lumen diameter of about 6 millimeters. An anti-coagulant is placedin the tube before the specimen is drawn. The specimen is taken by aneedle 159 (shown in broken lines in FIG. 8) which extends throughstopper 26 in a conventional manner, and such blood specimen tubes, asthus far described and blood specimen drawing techniques, are well knownin the medical profession.

While the present process is described by reference to preferredspecimen tube 21, it will be apparent from the following descriptionthat other forms of blood specimen containers can be used in the presentprocess.

In order to provide several important advantages, as will be set forthbelow and, particularly in order to provide a very thin cross-sectionalportion of the specimen in lumen 23, tube 21 preferably has an elongatedmember or rod 27 mounted in the lumen. Rod 27 is preferably about 4millimeters in diameter and is secured or held by adhesive, fused glassor other means, against one side of lumen 23, in this case the upwardlyoriented side 28.

In the preferred form, tube 21 has a substantially cylindrical lumen 23and elongated member 27 is a cylindrical rod, but other lumen-rodconfigurations are suitable for use in the present invention.

As set forth in the background, the Westergren lag phase can require 5to 15 minutes to complete. During this time rouleaux must be formed fromindividual erythrocytes in sufficient number to seed the specimen andstart the linear or decantation phase of sedimentation.

In the present invention rouleaux formation is induced at an acceleratedrate and sufficient rouleaux created to begin the decantation phase in amatter of seconds, for example, 30 seconds or less. The preferred mannerof accelerating rouleaux formation is to provide a specimen containerwhich will have a very thin cross-sectional area in one portion of lumen23. As will be seen in FIG. 5, the combination of cylindrical rod 27 andcylindrical lumen 23 is that two areas of small cross-section, namely,horn-like areas 31 and 32, exist proximate upper side 28 of lumen 23.Thus, blood specimen 29 in these areas is at least somewhat confined.While it may appear that horn-shaped cross-sectional areas 31 and 32 are"small" or "thin" in cross section, in the absence of lengthwise currentinduced by the exaggerated settling of rouleaux in the veils, they donot by themselves produce the accelerated rouleaux formation of themethod of the present invention.

As shown in FIG. 4, upper end 33 of rod 27 protrudes above top surface34 or the meniscus of specimen 29 for a tube oriented as shown, in thiscase, oriented at an angle of 30 degrees to a horizontal plane 36. Aswill be seen, the blood specimen forms an arcuate veil-like volume 37 ineach of horn areas 31 and 32 proximate and up to the upper end 33 of rod27. While impractical to show in the drawing, very thin specimen veil 37will extend up rod 27 by capillary action to the last point of contact38 of the rod with tube upper side wall 22. As arcuate specimen veil 37extends upwardly toward point 38, its cross section thins and in facttapers down to what is believed to be a relatively small multiple of thered cell diameter, for example, a veil cross section of 10 celldiameters (70-80 microns) or less. Thus, in the area of specimen veil 37red cells are forced to be in such close proximity to each other thatthey cluster and form rouleaux in a few seconds. For example,substantially all the red cells in the thinnest upper regions of veil 37are believed to be in rouleaux in 15 seconds.

Obviously, however, the amount of the specimen in the upper reaches ofveil region 37 is small as compared to the entire specimen 29. It isbelieved, however, that rouleaux are starting to form elsewhere in thespecimen and particularly in small cross-sectional horns 31 and 32 allalong the rod. This rouleaux formation is probably somewhat acceleratedas compared to formation in a Westergren tube, but it still would resultin an undesirably slow lag phase. Nevertheless, specimen 29 enters thedecantation or linear sedimentation phase almost immediately afterrouleaux formation has been completed in the upper end of veil region 37when specimen tube 21 is held in the 30 degree orientation shown in FIG.4.

It is hypothesized that the rouleaux formed in very thin upper region ofveil 37 are immediately free to gravitate downwardly along veil 37 andacross rod 27, and as they do so plasma is pulled up along hornedregions 31 and 32 between the rod and tube and into veil region 37. Theorientation of tube 21 is such that rouleaux can escape veil region 37and plasma can enter the veil region. There rapidly begins to occur,therefore, a circulation pattern in which denser rouleaux settle downthrough and "seed" the specimen picking up more cells as they fall andyet not trapping plasma below. The plasma, on the other hand rises upthe tube along horns and replaces the downwardly settling rouleaux. Asthe plasma rises, its motion is believed to cause free erythrocytes toagglomerate with partially formed rouleaux all along horns 31 and 32,thus causing further rouleaux formation and sedimentation.

The exact mechanics of acceleration are not known, but in a bloodspecimen of high sedimentation rate within about 30 seconds flow ofplasma upwardly along horns 31 and 32 toward veil 37 can be clearly seenin specimen 29 and rouleaux clearly has formed in an amount sufficientto cause the entire specimen to be in the decantation phase.

At the present time many aspects of this technique for acceleratingrouleaux formation are unknown. It is known, however, that the samespecimen tube assembly will require 15 minutes to complete thedecantation phase when rod 27 does not extend above specimen top surface34 and only 5 minutes when it does extend above surface 34. Thus, if thehorn regions 31 and 32 are used alone the specimen can be settled in 1/4the time of a Westergren sedimentation test, but if the specimen crosssection is further thinned by extending rod 27 beyond surface 34 to formveil region 37, the sedimentation time can be reduced to 1/12 of theWestergren sedimentation time.

It is believed that rod 27 should extend above upper specimen meniscusby at least 3 to 4 millimeters for optimum acceleration effect, but anyprotrusion starts to provide a thinned capillary veil which should beuseful. The maximum useful protrusion similarly is not known, but bloodspecimens will rise up to an inch or more above meniscus surface 34along thin capillary-like channels, such as horns 31 and 32.

As will be appreciated, various other specimen tube configurations arepossible to produce a very thin capillary cross section. Convergingplanar walls and converging planar and curvalinear walls may provide thesame effect, and such structures can be provided by lumen inserts orintegrally formed tube wall configurations. To achieve the best results,it is believed to be advantageous to employ capillary forces to form,with a portion of the tube lumen, a thin veil or film of the specimen.Such a veil or film, however, also must be positioned, it is believed,so that the rapidly formed rouleaux are free to escape film or veilregion 37 and seed the specimen, preferably while plasma is free toenter the film or veil portion of the lumen.

At the present time the orientation of tube 21 which is best suited forboth accelerated rouleaux formation and gravity settling during thedecantation phase is believed to be between about 20 degrees to about 35degrees from the horizon. Most preferably tube 21 is oriented at about30 degrees from horizontal plane 36 during both accelerated rouleauxformation and gravity settling during the decantation phase. As tube 21is lowered to an orientation below about 25 degrees, circulation ofplasma up along horns 31 and 32 is reduced and sedimentation times beginto increase. Similarly, as tube orientation is increased to above about35 degrees egress of plasma becomes impeded by settling of red cellaggregates which, at these higher angles no longer seed thesedimentation process but instead impede plasma egress.

Rapid Decantation and Syneresis Elimination

The next step in the process of the present invention is to gravitysettle the specimen, which is now through the lag phase ofsedimentation. The middle or decantation phase tends to be relativelylinear until the hematocrit increases significantly and the settlederythrocytes trap or impede upward migration of plasma at the bottom ofthe specimen tube, that is the specimen enters the syneresis phase.

In the present process, specimen container 21 is oriented in a mannerenabling settling of erythrocyte cells through upwardly migrating plasmawithout trapping or impeding of the plasma as a result of the relativelysmall transverse cross sectional area of the tube. Westergren tubes, andsettling tube 21, both have relatively small diameters in order that theamount of whole blood required to perform sedimentation testing can beminimized, but it is the small diameter which causes the slowing ofsedimentation at the end of the settling period. In the presentinvention the time required for the linear decantation phase is greatlyshortened and the syneresis phase is substantially eliminated byorienting specimen tube 21 with its longitudinal axis 55 of specimentube 21 in a near horizontal orientation, as shown in FIG. 4. As usedherein, "near horizontal" shall mean between about 35 degrees and aboutzero degrees from the horizon. Orienting longitudinal axis 55 in a nearhorizontal plane cause the transverse area of specimen tube 21 to begreatly increased as compared to the transverse area (FIG. 5) of avertically-oriented tube 21.

Such an orientation of the specimen tube 21 will cause the rouleaux inthe specimen tube to simultaneously gravitate or fall across therelatively small diameter of the tube, but over a relatively large areaof the tube. The resistance to downward movement of rouleaux and toupward migration of plasma in the tube as a result of the largehorizontal area of the near-horizontal, elongated tube substantiallyeliminates impeding, choking or slowing down of the sedimentation rate.The syneresis phase, as a result of dramatically increasing hematocritand resultant trapping of plasma by the erythrocytes, is substantiallyeliminated. By orienting the settling tube in a near horizontalorientation, the rouleaux move or settle down through the plasma in amanner which is very close to or approximates settling in a bottomlesstube. The hematocrit or blood cell packing density buildup has verylittle effect in trapping plasma because of the large transverse orhorizontal area and of the very short distance need for plasma travel toescape the settling erythrocytes.

Moreover, since the distance across the tube diameter is short, the timerequired for completion of the decantation or linear settling phase ismuch less. It has been determined that, after about 3.5 to about 5.0minutes of gravity settling with the specimen tube in a horizontalorientation, the decantation or linear sedimentation phase is completedin a degree which is substantially equal to, and correlatable with, 60minutes of settling using the Westergren method.

Sedimentation Determination

The next step in the process of the present invention, therefore, is todetermine the amount of settling which has occurred during the period oftime which the lag phase was accelerated and the decantation phase wastaking place, for example, 5.0 minutes. In order to facilitate adetermination of the amount of settling which has occurred, it ispreferable to reorient specimen container 21 to a near verticalposition. Thus, tube 21 can be rotated to a near vertical orientation,for example, to about 90 degrees from the horizon. This causes thesettled erythrocytes to slip down the bottom side of the specimen tube21 and the plasma on upper side of lumen 23 to float up the oppositeside of the tube to the top.

The reorientation of the specimen tube to a near vertical position willcause the settled cells to reach the bottom of the tube quickly, and ameasurement of the separation boundary or interface between the plasmaand settled cells can be taken, for example, as soon as five secondsafter reaching a vertical orientation. In the vertical orientation thelocation of the separation boundary between plasma and erythrocytes canbe more accurately determined than in the near horizontal orientation.The tube may, however, be read in a semi-vertical position with a modestincrease in associated reading error. As described above, the steps ofaccelerated induction of rouleaux formation, gravity settling in anorientation shortening the decantation phase and determining the amountof settling can be implemented by hand by a laboratory technician. Bysimply employing a rack or tube support structure which will hold tubes21 in the orientation of FIG. 4 and then a second rack which can be usedto vertically orient the tubes, a technician can easily perform theprocedure of the present invention without automation or specialequipment.

Semi-Automated Sedimentation Apparatus

Nevertheless, it is an important feature of the present invention thatthe method of the present invention can be easily semi-automated. FIGS.1 through 3 show one form of sedimentation tube manipulating apparatus20 which can be used to perform the present process. Sedimentationapparatus 20 preferably includes a base 41 having leveling assembly,such as manually engageable leveling screws 42 and a spirit level 43, sothat the apparatus can be leveled for reproducible results on alaboratory bench top. Mounted on base 41 is a housing 43 whichpreferably includes a translucent front panel 44 behind which a lightsource, such as two U-shaped fluorescent tubes 46 and 47 (FIG. 3), arepositioned.

In order to enable proper orientation and manipulation of sedimentationtubes 21 and 22, a rotatable tube holder assembly, generally designated48, is mounted to extend from front panel 44 of housing 43. In the formof sedimentation apparatus 20 illustrated, assembly 48 includes acentral U-shaped frame member 49 mounted by collar 51 to a shaft 52 forrotation therewith. Secured by fasteners to U-shaped frame member 49 aretube holder members 53 and 54. Each of the tube holder housings 53 and54 includes a slotted or windowed front opening 56 with sedimentationmeasuring indicia 57 positioned closely proximate thereto. As best maybe seen in FIG. 3, the back side 58 of housings 53 and 54 is open sothat light may enter the housing and fall upon tubes 21. Housings 53 and54 further include two mounting support felts (not shown) which receiveand firmly hold the tubes in the housings without interfering with thepassage of light through the tubes and out slots 56. As will be seenfrom FIG. 3, the tubes 21 are mounted in housings 53 and 54 with rod 27on the same side of the respective housings so that when assembly 48 isstopped in the position shown in FIG. 2, rods 27 will be oriented insidetubes 21 essentially as shown in FIG. 4.

Shaft 52 extends through front wall 44 of housing 43 and completelythrough the housing and out back wall 59. A first spur gear 61 ismounted on the end of shaft 52 and cooperatively engages a pinion 63carried by shaft 64 of motor 66. Mounted interiorly of housing 43 is anindexing disk 67 which is fixed for rotation with shaft 52 by collar 68.Also mounted interiorly of housing 43 is a magnet 69 and a ferromagneticcollar 71 coupled to shaft 52. Finally, an indexing detent assembly 72and light ballasts 73 also are mounted inside housing 43.

Operation of sedimentation apparatus 20 to implement the method of thepresent invention can now be described. In the preferred form, the firststep of the present invention is to thoroughly mix specimen 29 inspecimen tube 21. Mixing insures that there is no pre-formed rouleaux inthe specimen, and mixing thoroughly mixes the anti-coagulant material inthe specimen tube with the whole blood that has been drawn. Apparatus20, therefore, preferably is constructed in a manner which willmanipulate tube 21 so as to effect mixing.

As will be set forth in more detail below, one of the substantialadvantages of the construction of specimen tube 21 in which rod 27 ispositioned in lumen 23 is that the gas bubble which will always bepresent in the tube can be used as a mixing device. Thus, by invertingtube 21 the bubble will move from one end of the tube to the other, withthe blood/anti-coagulant moving in the opposite direction. One of thesubstantial problems in connection with mixing small liquid volumes isthat in small diameter tubes gas bubbles or the like will bridge, ratherthan move up and down the tube, and prevent mixing. The presence of therod in lumen 23 enables the blood/anti-coagulant to pass beyond thebubble in horn regions 31 and 32, because the bubble cannot enter intothe thin transverse cross section horn regions.

As a first step, therefore, motor switch 74 can be turned on and themotor 66 will slowly rotate the holder assembly 48 when the shaft andgears are in the solid line positions shown in FIG. 3. Thus, gear 61 isengaged with gear 63 and assembly 48 is positioned out away from frontpanel 44 of housing 43. The gearing and motor speed can be set so thatrotation occurs at about 3 rpm or 6 inversions per minute. About threeminutes of rotation will insure that the specimens in tube holderassembly 48 are thoroughly mixed.

In a semi-automated process the technician merely turns switch 74 offafter about three minutes of mixing. In a more fully automated process,termination of the mixing cycle is controlled by a timer. The gears 61and 63 can be retained in interengagement by magnetic member 69 whichattracts collar 71 thereto and maintains shaft 52 and gear 61 in thesolid line position of FIG. 3.

Once mixing is complete, the technician can push the shaft inwardly tothe dotted line position shown in FIG. 3, freeing collar 71 frommagnetic attraction of magnet 69 and freeing gear 61 from pinion 63. Astube holder assembly 48 is pushed inwardly towards panel 44, indexingdisk 67 also is moved to the dotted line position at which it is engagedby indexing detent assembly 72. Detent disk 67 can have two notcheswhich receive detent element 76 when the notches are in indexed relationto element 76. The indexing disk 67 is fixed by a collar for rotationwith shaft 52 and has a first notch at a location which will secure tubeholder assembly 48 in the position shown in FIG. 2, namely, at an angleof about 30 degrees to a horizontal plane. A second notch is provided inindexing disk 67 which will hold tube holder assembly 48 in the positionof FIG. 1, namely, in a near vertical position.

Accordingly, in a semi-automated system, the technician turns motorswitch 74 off, pushes tube holder assembly 48 inwardly to a positionclosely adjacent to translucent front panel 44 and rotates the assemblyto the position of FIG. 2, at which it is held in place by detentelement 76 engaging a notch indexing disk 67. The assembly is left inthis position for five minutes. The technician can then manually rotateassembly 48 from the FIG. 2 position to the FIG. 1 position, at whichpoint detent 76 will engage a second notch indexing disk 67, holding theassembly as shown in FIG. 1. The technician can then read the locationof the plasma/erythrocyte interface using indicia 57 on the front oftube holder housings 53 and 54.

One of the important and highly advantageous aspects of the presentinvention is that each blood specimen 29 can repeatedly have itssedimentation rate tested. Accordingly, the technician normally willcomplete the measurement process and then return the tube holderassembly to the FIG. 3 solid line position and turn on the motor tore-mix the specimen. It should be noted, of course, that light switch 77should be turned on so that reading of the plasma/erythrocyte interfaceor boundary can be easily accomplished by back lighting of the specimenthrough opening 58.

Using the apparatus of FIG. 1, therefore, a technician could easilyobtain six or seven sedimentation readings in the time period requiredto obtain one reading using the Westergren process, including the timerequired to re-mix the specimen after each sedimentation determination.

As will be appreciated, displacement of assembly 48 between the solidand dotted line position shown at FIG. 3 also can be fully automated,and it would also be possible to automate the determination of thelocation of the plasma/erythrocyte interface. Thus, automated opticalreading assemblies can be positioned proximate tubes 21 in moresophisticated systems so that the entire process, includingregistration/recording of sedimentation results can be automated.

FIG. 6 shows a graph of sedimentation results obtained using apparatus20 and the process of the present invention. These results have beencompared to Westergren sedimentation rates for the same specimens. Thespecimens are whole blood specimens which have been modified in thelaboratory to change their sedimentation rate in a manner well known andset forth in the blood sedimentation literature. As will be seen, usingapparatus 20 and the method of the present invention one can simplymultiply the results of use of the present apparatus by 2.0 over thefull range of sedimentation and obtain Westergren sedimentation rates.The best fit line for data taken using the present apparatus and methodis not significantly different from a line of identity with theWestergren rates, that is, the r² variation is equal to 0.98.

Specimen Container

One of the problems which is particularly acute with blood specimensedimentation studies is that it is highly desirable to minimize theamount of the specimen which is drawn. If a small volume of bloodspecimen is placed in an elongated specimen container, however, it isrelatively difficult to effect mixing of the specimen, even when dilutedwith anticoagulant. Long thin specimen containers with low volumes ofblood will not readily allow a bubble to migrate from one of thecontainer to the other. Even beads or balls are sometimes difficult toemploy as gravity mixing devices in small diameter elongated tubes andsuch mixing devices may damage red cells and cause a release ofhemoglobin.

In an additional broad aspect of the present invention, therefore, amethod and apparatus for mixing constituents of a small volume of liquidin a container having a small cross section is provided by the preferredform of specimen tube 21 and 121 of the present invention, as shown inFIGS. 4 and 8 of the drawings. Specimen tubes 21, 121 can be elongatedtubular members having a central lumen 23,151 extending along the lengthof the tube. An elongated member 27, 152 is mounted in the lumen andpreferably extends over a majority of the length of the lumen. Elongatedmember 27, 152 can be secured to an interior surface of the tube alongone side of the tube, for example, by an ultraviolet-activated adhesive153, or by other means, such as fusing a glass member to the interior ofa glass tube, or magnetically holding the rod to a side of the tube,which will be described in more detail hereinafter.

The mixing advantages of tube 21, 121 will be described by reference toFIGS. 8 and 9 and tube 121, but it will be understood that tube 21 issimilarly constructed. As best may be seen in FIG. 9, the specimen tubewall 150 and member 152 define therebetween a lumen 151 having atransverse cross section configuration which will prevent a gas bubblefrom completely filling the cross section of the lumen, which wouldprevent the liquid from passing beyond the gas bubble as it rises in thetube. This can be accomplished, for example, by providing a wedge-shapedtransverse area. As will be seen in FIG. 8, the use of a tube 121 havinga cylindrical bore with an elongated cylindrical rod 152 mounted thereinwill define therebetween a lumen 151 which is crescent-shaped intransverse cross section and has two wedge-shaped horn regions orconverging areas 156 at ends of the crescent.

When an air bubble 157 is present in a specimen tube constructed asshown for tube 121, surface tension forces will prevent the air bubblefrom extending into the converging wedge-shaped crescent ends 156 of thecross sectional area. As specimen tube 121 is tilted, therefore, bubble157 will migrate up the length of the tube in the widest portion of thecross section of crescent 154 while the liquid, blood and anticoagulant,will migrate down the tube past the bubble in the horn regions orwedge-shaped ends 156 into which the bubble cannot extend. Thus, even involumes as low as 1.2 milliliters in specimen containers having adiameter of only 6 millimeters, tilting of the specimen tube back andforth by sedimentation apparatus 120, or rotation by apparatus 20, willallow bubble 157 to be very effective in mixing the liquid constituentsin the container.

In the preferred form, therefore, specimen tube 121 are vacuum tubeswhich contains a predetermined amount of specimen diluent, such as,about 0.25 milliliters anticoagulant material, suitable for mixing witha 1.0 milliliter volume of whole blood. Such vacuum tubes withanticoagulant and a vacuum which will draw a predetermined known amountof blood specimen are well known in the art. These vacuum tubes do nothave elongated member 27, 152 mounted therein. The vacuum tube container21, 121 of the present invention, therefore, includes a rubber endstopper 26, 158 through which a needle 159 can be inserted. The innerend 161 of the needle preferably should not contact the outermost end162 of member 152. Thus, end 162 of member 152 is recessed by an amount(for example, one-half centimeter) to allow inner end 161 of needle 159to clear member 152.

Since it is not possible to draw a perfect vacuum inside specimen tube121, there will always be some gas, usually air, trapped in the vacuumtube. When a specimen is drawn, therefore, it will be pulled into lumen151 by the vacuum therein until the lumen between the tube and elongatedmember is substantially filled (about 1.25 milliliters), with theexception of a small air bubble 157. Air bubble 157 can then be used tomix liquid after needle 159 is removed from rubber stopper 158.

A major advantage of specimen container 21, 121 and the method andapparatus of the present invention is that at all times the bloodspecimen is sealed in container 21, 121. Thus, the danger to techniciansfrom handling whole blood is greatly reduced. Moreover, the samespecimen can be used in the sealed container to repeat the sedimentationprocess. As will be seen from FIG. 8, specimen tube 121 includes astopper 58 having a diameter greater than the tube body. This willresult in a slight tilt to the specimen tube, for example, of 2-4degrees, from the horizon when tube 121 is placed in a horizontallyoriented trough 137. This has the advantage that it ensures that bubble157 will be up at stopper 158 so that the bubble will not cause remixingwhen the tube is reoriented to a near vertical orientation to determinethe amount of settling. It is a feature of the present process,therefore, to effect gravity settling with container 121 oriented withupper end or stopper 158 tilted up from horizontal by about 1 degree toabout 6 degrees.

Referring now to FIG. 9A, still a further alternative embodiment 121a ofthe specimen container of the present invention is shown. An elongatedplastic insert member 181 is positioned in lumen 151a of tube 121a. Theplastic insert includes horn regions or wedge-shaped cross sectionalareas 156a into which a gas cannot extend. Thus, the liquid in tube 121awill pass beyond the gas bubble in areas 156a as the tube is tilted. Itis believed that a tube constructed as shown in FIG. 9A, if orientedwith one horn region 156a uppermost, and if a very thin section extendedupwardly beyond the specimen upper surface 34, would accelerate rouleauxformation and be suitable for use in apparatus 21.

Still a further alternative tube embodiment is shown in FIG. 9B. Tube121b has been formed to provide the narrow cross sectional areas 156bby, for example, deforming a heated glass tube to provide narrow areas156b. Again, areas 156b are sufficiently narrow to prevent a gas fromentering these areas, and the liquid can move past the bubble in theseareas. Whether or not glass fabrication techniques will allow asufficiently thin horn region 156 to be formed to allow use of theaccelerated rouleaux formation technique described in connection withFIGS. 4 and 5 is unknown.

First Alternative Lag Phase Acceleration Process and Apparatus

While the preferred form of the method and apparatus of the presentinvention have been described in connection with FIGS. 1-6, it also hasbeen discovered that there are alternative ways for accelerating the lagphase of blood sedimentation, and the principles of shortening thedecantation phase by orienting the specimen tube in a near horizontalposition can be combined with these various alternative lag phaseshortening techniques. Such alternative techniques are believed to havedisadvantages in connection with the apparatus which implement them, butthey do produce accurate data in a short time which can be linearlycorrelated to Westergren sedimentation rates. Accordingly, for someapplications, these alternate embodiments may have certain advantages.

Referring now to FIG. 7, an alternate sedimentation apparatusconstructed in accordance with the present invention is shown.Sedimentation apparatus 120 preferably employs a centrifuge assembly asa means for inducing rouleaux formation in the blood specimen. Thus,centrifuge assembly 122 includes a rotatable turntable 124 on which tubereceiving head 126 is mounted. A plurality of elongated tube receivingnotches 127 are formed in head 126 and a tube retaining means, such asbrand 128, can be mounted over head 126 so as to retain specimen tubes121 in notches 126 during rotation of the centrifuge. Various otherforms of tube retaining structures are suitable for use with centrifuge122. Turntable 124 is mounted to a drive controller assembly 129. Powercan be controlled through on-off switch 131, the spin rate by controlknob 130 and the spin duration through knob 135.

Centrifuge assembly 122 of the present invention can be provided by anyone of a number of standard laboratory centrifuges as long as they arecapable of spinning elongated specimen tubes 121 with the longitudinalaxis thereof generally parallel to the spin axis of the centrifuge at arate high enough to induce rouleaux formation in an amount sufficient tocause erythrocyte settling to begin at substantially the decantationrate for the specimen in a short period of time.

Centrifuge assembly 122 accelerates the specimen with a centrifugalforce that preferably is in the range of about 5 to 10 times theacceleration of gravity, g. A centrifuge operating at about 400 rpm witha distance from the spin axis to the center of the specimen tube ofabout 4 centimeters will produce sufficient centrifugal force to causethe somewhat more dense erythrocytes to migrate through the plasma tothe outermost surface defining the specimen tube bore or lumen. As theerythrocytes are driven to the tube side, they come in contact with eachother and begin to adhere together to form rouleaux which are heldagainst the outer side of the lumen by the centrifugal force. Afterabout 20 to 45 seconds, sufficient rouleaux will be formed on the outerside of the specimen tube lumen so that, if the rouleaux are allowed togravitate, they will begin settling of the specimen at the decantationrate.

As above described, the next step in the process of the presentinvention is to gravity settle the specimen, which is now through thelag phase of sedimentation. Specimen container 121 again is oriented ina manner for settling of erythrocyte cells through upwardly migratingplasma without being constricted or impeded by the relatively smallcross sectional area of tube 121. This can be accomplished using thepreferred elongated, small diameter specimen tube by placing thespecimen tube in manipulation apparatus 123 with the longitudinal axis155 of specimen tube 121 in a near horizontal orientation, in this caseessentially zero degrees or in a horizontal plane, as shown in FIGS. 7and 8. Orienting axis 155 in a substantially horizontal plane cause thetransverse area of the specimen tube to be greatly increased as comparedto a vertically-oriented tube 121. Additionally, the outer side 160 ofthe specimen tube, against which the rouleaux were formed bycentrifugation, is oriented in an uppermost position. Thus, specimentubes 121 are transferred from centrifuge head 126 to a support member136, which has a plurality of tube-receiving mounting structures, suchas troughs or grooves 137. Specimen container 121 is oriented with itslongitudinal axis 155 horizontal and side 160, which was facingoutwardly in the centrifuge, facing upwardly on tube support member 136.This results in rod 152 in tube 121 being on bottom side 150 of thetube, which is just opposite of the position of rod 27 in tube 21 duringdecantation.

Again, the distance across the tube diameter is short, and the timerequired for completion of the decantation or linear settling phase ismuch less than for the Westergren process. After about 3.5 minutes ofgravity settling the specimen tube 12 in a horizontal orientation, thedecantation or linear sedimentation phase is completed to a degree whichis substantially equal to, and correlatable with, 60 minutes of settlingusing the Westergren method.

In order to facilitate a determination of the amount of settling whichhas occurred, it is preferable that sedimentation apparatus 120 reorientspecimen container 121 to a near vertical position. Thus, over a timeinterval of approximately 15 to 40 seconds, support member 136 can berotated to a near vertical orientation, for example, to about 80 degreesfrom the horizon. This causes the settled erythrocytes to slip down thebottom side of the specimen tube 121 and the plasma on upper side 160 ofthe lumen 151 to float up the opposite side of the tube to the top.

It has been found that reorientation of the specimen tube 121 to a nearvertical position must be accomplished more slowly than reorientation oftube 21 in order to avoid re-mixing of settled cells. Once in thevertical orientation, however, the location of the separation boundarybetween plasma and erythrocytes can be accurately determined using scale138 next to troughs 137.

As will be apparent from FIG. 7, container orientation assembly 123preferably includes support axle 139 and control and motor assembly 141which can be used to rotate tube support member 136 from a horizontal toa near vertical position in a slow but smooth reorienting step.Apparatus 123 also may include means (not shown) for retaining each oftubes 121 in troughs 137 during their orientation to a near verticalposition, although since they do not go beyond vertical, tubes 121 canbe retained in troughs 137 by means of gravity.

In the method of the present invention, it is preferable that the stepof mixing the blood specimen be undertaken immediately prior to inducingrouleaux formation. Thus, sedimentation apparatus 120, and particularlycontainer-orienting apparatus 123, of the present invention preferablyis formed to mix the blood specimen thoroughly just prior to thecentrifuging or rouleaux inducing step.

Sedimentation apparatus 120 preferably includes a controller 141, whichincludes a mixer input 142 that will cause tube support member 136 tooscillate about a horizontal axis, for example, about axle 139. Suchoscillation or tilting can be used to cause a gas bubble or a glass bead(not shown) to migrate from one end of specimen container 121 to theother. It would also be possible to effect mixing by a continuingrotating process, but then retention means for the specimen containersclearly would be required. In the preferred form, an air bubble in thespecimen container is allowed to migrate from one end of the containerto the other by tilting specimen container 121 by about 60 degrees to 75degrees from the horizon in one direction and then by about the sameamount from the horizon in the opposite direction. Such tilting can takeplace at about six inversions or full swings per minute forapproximately two minutes. The number of tilt cycles can be adjusted byknob 145.

Referring now to FIGS. 10 through 14, details of the process of thepresent invention and operation of the sedimentation apparatus 120 canbe more fully described.

FIG. 10 schematically illustrates specimen tube 121 immediately afterobtaining a specimen of whole blood and removal of needle 159. Container121, a 110 millimeter long tube with a 6 millimeter internal diameterbore and a 4 millimeter diameter rod in it, is filled with whole bloodand diluent 171, as well as an air bubble 157, which represents the airleft in the incompletely evacuated specimen tube. Specimen tube 121 isplaced in tube orienting apparatus 123 in one of the grooves or troughs137 in tray 136. The technician then can turn on the orientation device123 by switching switch 140 to start the mixing process. Mixer light 142will come "on" and the specimen tube is tilted about axle 139 above andbelow the horizon by about 70 degrees, as schematically illustrated inFIG. 11. Bubble 157 migrates through specimen 171 along the member 152as the liquid specimen passes beyond the bubble in the crescents createdby the rod 152 positioned in bore 151 of tube 121. The mixing process iscontinued at six inversions per minute for approximately 2 minutes, atwhich time controller 141 for the mixer brings tube supporting tray 136to a horizontal stationary position and turns off mixing light 142. Itmay be necessary for controller 141 to slow the rate of tilting or evenstop at the extremes of the cycle to allow effective mixing,particularly of contents proximate the tube ends.

The technician then lifts tube 121 from tray 136 and places the same inone of the notches 127 of centrifuge head 126. The operator turns thecentrifuge "on," using actuator button 131, and the tube will be spunabout a spin axis 172, as schematically illustrated in FIG. 12. The spinrate will typically be 400 rpm at a radius 173 of 4 centimeters.Centrifugation continues for approximately 20 seconds, which will causea layer of rouleaux, schematically illustrated at 174, to form along theoutermost side 160 of tube bore 151. Bubble 157 will be present at thetop of the tube, and the tube will be oriented in centrifuge head 126with the rod 152 closest to spin axis 172 so that the rouleaux will beinduced to form and collect against the tube wall farthest from rodmember 152.

Once the centrifuge step is completed, the technician will removespecimen tube 121 from centrifuge 126 and place the same in troughs 137of tube orienting assembly 123, namely, in a near horizontal orientationas shown in FIG. 13. Preferably, there is no more than about 6 degreesupward tilt of the stopper end induced by a combination of stopper 158and the trough orientation. Some upward tilt is advantageous in that itensures that air bubble 157 will be located just under the stopper. Itwill be seen from FIG. 13 that rouleaux layer 174 and side 160 which wasoutermost in the centrifuge will be oriented in the uppermost position.Conversely, rod 152 is now in a lowermost position. Air bubble 157 willbe located at the top of the tube under stopper 158. The technician canthen press actuator button 171, and the tray orienting controller 141will hold the tray in a generally horizontal position for approximately3.5 minutes.

FIG. 13A shows the specimen in tube 121 at the start of gravitysettling. Rouleaux 174 can be seen to be collected proximate theuppermost side 160 of the tube and the remainder of the specimen 177 iscomprised of a mixture of erythrocytes and plasma in suspension. Thecentrifugation step, however, has created sufficient rouleaux that uponplacement of the tube in the horizontal position on tray 136 rouleauxwill begin to settle or gravitate downwardly through mixture 177 ofplasma and red blood cells. In a manner analogous to cloud seeding, asthe rouleaux begins to fall through mixture 177, additional erythrocytesadhere to the falling clusters and additional rouleaux are formed. Thespecimen undergoes the relatively linear decantation phase in whichsettling or sedimentation occurs relatively rapidly.

At the end of a predetermined gravity settling period, for example, 3.5minutes, the specimen will have the appearance as schematicallyillustrated in FIG. 13B. The bottom of the crescents of lumen 154 willbe filled with sedimented erythrocytes, largely in rouleaux 174. Amiddle area of the specimen will contain a mixture 177 of plasma anderythrocytes in which rouleaux are less densely packed. Finally, a layerof plasma 178 will be present proximate uppermost side 160 of specimentube 121. A separation boundary 179 also will be present between plasma178 and the remainder of the specimen including erythrocytes.

The final step in the method of the present invention, therefore, is tomeasure the amount of settling which has occurred during the gravitysettling period. This can be theoretically accomplished by measuring thelocation of separation boundary 179 while the specimen tube is stillhorizontally oriented. As a practical matter, however, determination ofthe precise quantity of settling is less accurate when specimen tube ishorizontally oriented. Thus, it is preferable that the step ofdetermining the amount of settling be accomplished by reorienting thespecimen tube to the position shown in FIG. 14, namely a near verticalorientation. Such reorientation is accomplished automatically after 3.5minutes of gravity settling by controller 141, which smoothly andgradually tilts tray member 136 to a near vertical position, forexample, to 80 degrees above the horizon. As the specimen tube istilted, the rouleaux layer 174 sinks to the bottom end of the tube asdoes the mixture layer 177, while the lighter plasma layer 178gravitates to the top and comes to rest just under bubble 157. Theposition of separation boundary 179 can now be measured by comparing thesame to a measuring scale 138 on tray 136, or as shown in FIG. 13, ameasuring scale 138a on specimen tube 121, to determine the quantity ofsedimentation which has occurred during the centrifugation and gravitysettling time period.

The entire process, as above described, can be accomplished in less than5 minutes. Moreover, the quantity of sedimentation which has occurredcan be linearly related to Westergren sedimentation units by simplymultiplying the sedimentation result in millimeters of fall by a linearmultiplier. Using a best fit analysis of the data, extremely highcorrelation with Westergren data can be obtained using the apparatus ofFIG. 7 and a multiplier of 1.88. Correlation using a 1.88 times themeasured sedimentation values using the FIG. 7 apparatus and Westergrensedimentation values from specimens from the same patient have beenfound to have a Pearson correlation coefficient, r, value ofapproximately 0.99 and r² value of 0.95, which is an extremely highcorrelation.

Moreover, and very importantly, the same specimen can be tested againusing the present process by simply turning on the mixer after thesedimentation has been measured to re-mix and re-suspend theerythrocytes. The process is then run again until a new sedimentationvalue is measured. Repeated runs on the same specimen allow an averagevalue to be used. In 20-30 minutes, therefore, the same specimen can betested three times to produce a highly accurate Westergren sedimentationrate. Achieving three sets of sedimentation data using the Westergrenmethod would require 3 hours, if it could be done, which is notcurrently possible because the Westergren tube is not sealed, and thespecimen would have to be removed, remixed and reinserted into the tube,which would inevitably result in some loss of specimen, requiring theaddition of new specimen to make up for the loss. Additionally, such aprocess is extremely tedious and would require potentially dangerousexposure to contact with the specimen.

FIGS. 15 and 16 illustrate the high correlation of the sedimentationprocess of the present invention using the apparatus of FIG. 7 with theWestergren process. In FIGS. 15 and 16, the acronym "SSR" stands for"Speedy Sedimentation Rate" and indicates the process of the presentinvention. The acronym "ESR" stands for "Erythrocyte Sedimentation Rate"and indicates data taken using the Westergren process. FIG. 15 is basedupon blood specimens from patients known to have disease or inflammatoryconditions. FIG. 16 is based upon blood specimens taken from healthypatients, which specimens were modified, as is well known in the art, bythe addition of various amounts of gelatin and/or saline solution toincrease the sedimentation rate of the specimen to simulate thesedimentation rates which would occur when a disease or inflammatorycondition is present.

In FIGS. 15 and 16, the same blood specimen from a single patient wasdivided into two sub-specimens and sedimentation was measured with onesub-specimen using the Westergren process and the other sub-specimenusing the present process. In the present process, a centrifugation of400 rpm on a 4 centimeter radius for a time period of 20 seconds and agravity settling time of 3.5 minutes was used in each case. The SSR datawas compared to the ESR data and a multiplier of 1.88 was used in bothFIGS. 15 and 16 with the SSR data, based upon a "best fit" analysis ofthe SSR data to the line of identity.

In FIGS. 15 and 16, therefore, each data point represents twosedimentation rate measurements. Data point 91 on FIG. 15, for example,is a SSR measurement times 1.88 which yielded a sedimentation value of26 millimeters while the Westergren ESR value for the same patient was29 millimeters. Data point 92 is a SSR×1.88 value of 48 millimeters anda Westergren ESR of 39 millimeters.

Second Alternative Lag Phase Acceleration Process And Apparatus

A second alternative embodiment of the lag phase acceleration process ofthe present invention has been found to achieve accelerated bloodsedimentation rates which can be linearly related to Westergren rates.FIG. 17 illustrates an apparatus suitable for use with this embodimentof the present process.

A specimen tube 221 is provided which includes an elongated lumen 222having an elongated member 223 mounted therein. Member 223, however, isa ferromagnetic member, such as a glass or plastic tube 224 having aferromagnetic material 226 inserted therein and end seals 227. Mountedproximate tube 221 is an electromagnet device 228, and the specimen tube221 is supported on horizontal surface 229, although stopper 231provides a slight inclination so that bubble 232 is proximate thestopper end of the specimen tube.

As described above, the specimen 233 is first thoroughly mixed, forexample by tilting or rotating tube 221 about a horizontal axis to causebubble 232 to migrate from one end of the tube to the other. Duringmixing, if in a magnetized holder, the ferromagnetic rod is fixed to onewall and, if not, ferromagnetic rod 223 is free to translate from sideto side inside lumen 222. After mixing tube 221 is placed on surface 229and the electromagnet energized to pull member 223 to the upper side 235of lumen 222. The rod is held against upper side 235 for 20 seconds, andit is believed that during that time period rouleaux begin forming inthe horn regions between the rod and tube. If this technique were usedalone, however, rouleaux formation would still be undesirably slow.

After about 20 seconds, electromagnet 228 is turned off andferromagnetic rod 223 drops from upper side 235 of the tube lumen to thelower side 240 of lumen 222. The rod motion has a stirring or mixingeffect, but it is hypothesized that this motion of rod 223 throughspecimen 233 is sufficient to create further rouleaux and effectivelystart the decantation phase.

After 3 to 4 minutes of gravity settling, the specimen can be slowlyraised to a vertical position and sedimentation measured. Onedisadvantage of this alternative embodiment of the present invention isthat erythrocytes will remain in suspension in the plasma to a degreegiving the plasma a cloudy appearance. This may be the result of therod's motion through the specimen. Nevertheless, with strongbacklighting the plasma/erythrocyte interface can still be located andthe sedimentation rate determined. It is believed that location of theinterface may be most accurately determined by using automated opticalreading apparatus.

Sedimentation rates which linearly correlate to Westergren rates havebeen obtained in only 4 minutes using this modified embodiment of theinvention. As will be appreciated the advantages of shortening thedecantation phase by settling across the settling tube are againemployed, and syneresis is essentially eliminated.

In describing the preferred embodiments of process and apparatus of thepresent invention, it will be understood that many of the parameters canbe varied within the scope of the present invention. More particularly,gravity settling time periods of 3 to 5 minutes have been found toproduce settling rates in a 100 millimeter tube which merely requiresmultiplication by a factor of 1.75 to 2.25 to produce equivalentWestergren sedimentation rates with an extremely good correlation. Aswill be appreciated, however, shorter and longer gravity settling timescan be employed. Similarly, in the centrifugation embodiment shorter orlonger centrifugation times, and higher or lower centrifugation forces,can be employed in combination with differing linear multipliers.

For example, based upon somewhat limited specimen numbers using thecentrifugation process to accelerate rouleaux formation, it appears thatthe best fit multiplier for a 3.0 minute gravity settling period forcorrelation with Westergren sedimentation rates is a multiplier of 2.12in a 100 millimeter tube. Similarly, a 4.0 minute gravity settling stepappears to correlate with Westergren sedimentation rates using a 1.80multiplier. The most significant aspect of all three embodiments of thepresent process and apparatus is not the exact multiplier value, butthat the relationship is linear so that a single multiplier can be foundfor the present process once the various duration parameters are set. Itwill be appreciated, however, that as the length of the gravity settlingstep is increased, the erythrocyte packing density can become a factordriving the multiplier into a non-linear range. Thus, the preferred timeperiod for the gravity settling step in a 6 millimeter by 100 millimetertube with a 4 millimeter rod insert member is between about 2 to about 6minutes.

Changes in the length and diameter specimen container 21 are alsopossible and have somewhat less of an effect on the measuredsedimentation rates. Since settling occurs when the tube is on its side,the length dimension has very little effect, other than increasing theamount of blood that must be drawn. Diameter effects are greater, bothin terms of increasing the specimen size and the distance over whichplasma must upwardly migrate as the red cells settle. A lumen heightbetween the rod and tube when the tube is horizontal in the range ofabout 1 millimeter to about 6 millimeters is believed to be optimum. Thevolume of blood, however, will increase very rapidly with diameterincreases.

With respect to length of time of mixing, certain minimum mixing isrequired and there is no downside to additional mixing within reasonabletime limits such that the cells in the blood specimen are not damaged.There is, however, an overall increase in the time to process aspecimen.

The centrifuge time will effect results significantly. As the time isincreased, more rouleaux than would be formed during the lag phase willbe formed. Settling would accordingly be too rapid or cause thesedimentation multiplication factor to change. A maximum of about 45seconds of centrifugation can be tolerated, but 20 to 30 seconds at 5-10g's is preferred. As the spin radius and spin rate are changed, thecentrifugal force also will be varied, which will increase or decreasethe amount of rouleaux formation.

The rate of the reorientation step to enable measurement is notsensitive in the thin veil embodiment and only slightly more sensitivein the centrifugation and magnetic rod embodiments. If it is too fast,mixing can occur and if it is too slow, additional settling occurs.Reorientation in the range of about 2 to 4 seconds is permissible forthe thin veil embodiment and in about 15 to 45 seconds for thecentrifugation and magnetic rod embodiments produces substantially thesame results, with 20 seconds being preferred.

Once reorientation is accomplished, the sedimentation measurement can bemade almost immediately, for example, within 5 seconds. Waiting too longwill have some effect on the correlation of data, even though thespecimen hematocrit will be high as the specimen will be in the final orsyneresis phase. The effect of waiting too long to measure can besignificant because of the continual slumping of erythrocytes.

What is claimed is:
 1. A method of mixing the constituents of a smallvolume of liquid in an elongated tube by means of a bubble movinglongitudinally therein comprising the steps of:placing a small volume ofliquid to be mixed in an elongated tube having a lumen with a crosssectional configuration extending substantially over length of said tubepreventing entry of a bubble formed by a small volume of gas in saidtube into a portion of said cross sectional area of said lumen; andmoving said tube to cause migration of said bubble along said length ofsaid tube while said liquid passes beyond said bubble in said portion ofsaid cross sectional area of said lumen.
 2. A method as defined in claim1 wherein,said placing step is accomplished by placing said small volumeof liquid into a tube having a rod mounted in said lumen, said rodhaving a smaller external transverse dimension than the internaltransverse dimension of said lumen and said rod and said tube definingan elongated volume having a wedge-shaped transverse cross sectionalarea therein; and said moving step in accomplished by tilting said tube.3. A method as defined in claim 1 wherein, said placing step isaccomplished by placing said small volume of liquid into a lumen of atube having a side thereof inwardly deformed to define said crosssectional configuration.
 4. A method of mixing the constituents of asmall volume of liquid comprising the steps of:placing a small volume ofliquid into a lumen of a small diameter elongated vacuum tube under anapplied vacuum, said vacuum tube having an elongated rod mounted in saidlumen, and said rod and said vacuum tube defining a narrow transversecross sectional area in a portion of said lumen extending longitudinallyin said lumen and formed to prevent entry of a bubble of gas in saidvacuum tube into said narrow transverse cross sectional area of saidlumen; and moving said vacuum tube to cause migration of said bubblebetween opposite ends of said vacuum tube while said liquid passesbeyond said bubble in said narrow transverse cross sectional area ofsaid lumen, therein causing the effective mixing of said constituents ofsaid liquid volume.
 5. The method as defined in claim 4 wherein,saidstep of placing a small volume of liquid is accomplished by placing ablood specimen in said lumen of said vacuum tube.
 6. The method asdefined in claim 4 wherein,said placing step is accomplished by placingsaid small volume of liquid into a vacuum tube having a cylindricallumen with a cylindrical rod mounted therein; and said moving step isaccomplished by tilting said vacuum tube.